Methods and antibodies for designing and detecting mutation-specific or hidden epitope/antigena

Abstract

This invention discloses “Artificially Cleaved Epitope (ACE)” methods, antibodies, reagents, immunoassays, and kits for designing and detecting mutation-specific epitopes/antigens. The ACE methods can detect epitopes that are either absent or poorly recognizable or accessible naturally to antibodies, and thus must be specifically and artificially created (free terminals) and/or exposed in samples and sample preparations for antibody detection. The ACE methods comprise ACE antigen design and ACE antigen detection. The ACE methods, antibodies, reagents, immunoassays, and kits are useful in research and discovery, diagnostic, and therapeutic applications. In another aspect, the ACE methods can artificially and specifically expose hidden antigens while reducing the antibody non-specific bindings in all antibody-based applications.

We claim: 
     
       1. A method of detecting a mutation-specific or non-mutation-specific hydrolysis-created Artificially Cleaved Epitope (ACE) structure in a sample, wherein the ACE structure is present only in either a mutant form or the non-mutant form, comprising steps of:
 (i) designing an ACE structure derived from a basic mutation-specific structure H2N-A3-A2-A1-Am(s)-A1′-A2′-A3′-COOH, or a basic non-mutation-specific structure H2N-A3-A2-A1-An(s)-A1′-A2′-A3′-COOH, wherein A1, A2, A3, Am(s) or An(s), A1′, A2′ and A3′ are amino acid residue(s) of a polypeptide/protein, wherein Am(s) is the mutated amino acid residue(s) that is different with the corresponding non-mutated amino acid residue(s) or An(s), wherein the peptide bond(s) between Am(s) or An(s) and its adjacent amino acid residues is artificially cleaved via chemical bond-specific hydrolytic enzyme or agent, and wherein the antibody specifically recognizes at least the Am(s) or An(s) residue(s); 
 (ii) synthesizing the ACE structure; 
 (iii) making an antibody against the ACE structure; 
 (iv) creating the ACE structure in the sample by treating the sample preparation with the hydrolytic enzyme or hydrolytic agent, thereby exposing the formerly less antigenic or hidden ACE structure to specific interaction with the antibody; and 
 (v) detecting the ACE structure created in step (iv) with the antibody, wherein the antibody specifically binds to at least one of the artificially created Am(s) or An(s) residue(s). 
 
     
     
       2. The method of  claim 1  wherein the antibody is a polyclonal antibody, a monoclonal antibody, a bi-specific antibody, a recombinant antibody, a humanized antibody, a single chain or single domain antibody, an antibody fragment, or an antibody-like molecule. 
     
     
       3. The method of  claim 1  wherein the hydrolytic enzyme is selected from the group consisting of a protease, a glycosidase, a lipase, a phospholipase, a nuclease, a polyribosyl hydrolase, and their combinations. 
     
     
       4. The method of  claim 1  wherein the hydrolytic agent(s) is selected from a substance with chemical bond-specific hydrolysis activity. 
     
     
       5. The hydrolytic agents of  claim 4  wherein the hydrolytic agents are BNPS-skatole (3-bromo-3-methyl-2-[(2-nitrophenyl)thio]-3H-indole), CNBr (cyanogen bromide), formic acid, hydroxylamine (NH 2 OH), iodosobenzoic acid, and NTCB+Ni (2-nitro-5-thiocyanobenzoic acid). 
     
     
       6. The method of  claim 1  wherein the sample preparation is selected from the group consisting of a blot membrane, a tissue section, an isolated organ, an isolated cell, an isolated organelle, isolated tissue, an isolated body fluid, cell culture media, cell lysate, tissue lysate, an isolated fraction, a subcellular fraction, a chromatographic fraction, an immunocomplex, and a centrifuge fraction. 
     
     
       7. The method of  claim 1  wherein the step of synthesizing the ACE structure further comprises treating the synthesized ACE structure with a fixative. 
     
     
       8. The method of  claim 7  wherein the fixative is selected from the group consisting of an aldehyde, an alcohol, acetone, and osmium tetroxide. 
     
     
       9. A method of detecting a mutation-specific or non-mutation-specific hydrolysis-created Artificially Cleaved Epitope (ACE) structure in a sample, wherein the ACE structure is present only in either a mutant form or the non-mutant form, comprising steps of:
 (i) designing an ACE structure derived from a basic mutation-specific structure H2N-A3-A2-A1-Am(s)-A1′-A2′-A3′-COOH, or a basic non-mutation-specific structure H2N-A3-A2-A1-An(s)-A1′-A2′-A3′-COOH, wherein A1, A2, A3, Am(s) or An(s), A1′, A2′ and A3′ are amino acid residue(s) of a polypeptide/protein, wherein Am(s) is the mutated amino acid residue(s) that is different with the corresponding non-mutated amino acid residue(s) or An(s), wherein the peptide bond(s) between Am(s) or An(s) and its adjacent amino acid residues is artificially cleaved via chemical bond-specific hydrolytic enzyme selected from the group consisting of a protease, a glycosidase, a lipase, a phospholipase, a nuclease, a polyribosyl hydrolase, and their combinations or hydrolytic agent selected from a substance with chemical bond-specific hydrolysis activity, and wherein the antibody specifically recognizes at least the Am(s) or An(s) residue(s); 
 (ii) synthesizing the ACE structure; 
 (iii) making an antibody against the ACE structure, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a bi-specific antibody, a recombinant antibody, a humanized antibody, a single chain or single domain antibody, an antibody fragment, or an antibody-like molecule; 
 (iv) creating the ACE structure in a sample by treating the sample preparation with the hydrolytic enzyme or hydrolytic agent, thereby exposing the formerly less antigenic or hidden ACE structure to specific interaction with the antibody; and 
 (v) detecting the ACE structure created in step (iv) with the antibody, wherein the antibody specifically binds to at least one of the artificially created Am(s) or An(s) residue(s). 
 
     
     
       10. The method of  claim 9  wherein the sample preparation is selected from the group consisting of a blot membrane, a tissue section, an isolated organ, an isolated cell, an isolated organelle, isolated tissue, an isolated body fluid, cell culture media, cell lysate, tissue lysate, an isolated fraction, a subcellular fraction, a chromatographic fraction, an immunocomplex, and a centrifuge fraction.